RHODOSPIN GENE CORRECTION BY OLIGONUCLEOTIDE TARGETING

描述(改编自申请人的摘要)：三重导演和其他 将测试基于寡核苷酸的策略的可行性，因为 常染色体显性遗传性视网膜色素变性(ADRP)的基因特异性治疗 由视紫红质基因缺陷引起。在这项工作的过程中 研究人员将开发可用于测试各种 ADRP和其他常染色体显性遗传的治疗方法 精神错乱。因为任何治疗adrp的方法最终都必须在 对于动物，研究人员之所以选择老鼠，是因为它们的 基因组是可以修改的。研究人员提议创造一种小鼠胚胎干细胞 可有效靶向的DNA的线条可选择片段 特定部位重组。这些改良的ES细胞将允许高效的， 任何形式的视紫红质、c DNA或基因组、野生型或 突变体，来自任何物种，用于广泛的生理研究。这个 研究人员将“敲入”人类视紫红质基因，旨在为 作为寡核苷酸治疗影响的灵敏细胞探测器 视紫红质基因的表达、纠正和敲除。对于整个老鼠的研究， 研究人员将使用视紫红质与绿色荧光蛋白的融合 (GFP)。寡核苷酸对转录的影响将作为减少来衡量 在荧光强度上；寡核苷酸对基因校正和 突变将通过GFP荧光的出现来衡量，从 未表达的视紫红质-GFP融合构建体(GFP-)。细胞研究 将先于整个动物实验，以测试各种治疗方法 参数。尽管寡核苷酸介导的转录效应可以是 易于测量，基于重组的校正和突变检测 基因敲除需要专门的构造，调查人员最初将 中国仓鼠选择性腺苷磷酸核糖转移酶基因检测 细胞。灵敏的检测系统将使我们能够检测到微弱的信号 然后才能得到改进和优化。使用CHO寡核苷酸(TFOS)、RNA/DNA 嵌合寡核苷酸及其类似物，如肽核 酸(PNA)，因为它们有能力抑制转录，纠正基因缺陷， 并引入突变。这些结果将被用作开发 小鼠的治疗方案。

DESCRIPTION (Adapted from the applicant's abstract): Triplex-directed and other oligonucleotide-based strategies will be tested for feasibility as gene-specific therapies for autosomal dominant retinitis pigmentosa (ADRP) caused by defects in the rhodopsin gene. In the course of this work the investigator will develop technologies that can be used for testing a variety of therapeutic approaches to ADRP and other autosomal dominant genetic disorders. As any therapy for ADRP must ultimately be tested and optimized in animals, the investigator has chosen mice because of the ease with which their genomes can be modified. The investigator proposes to create a mouse ES cell line selectable segment of DNA that can be efficiently targeted by site-specific recombination. These modified ES cells will permit efficient, selectable 'knock-in' of any form of rhodopsin, cDNA or genomic, wild-type or mutant, from any species, for a broad range of physiological studies. The investigator will "knock-in" modified human rhodopsin genes, designed to serve as sensitive cellular detectors of the effects of oligonucleotide treatments on rhodopsin gene expression, correction and knockout. For whole mouse studies, the investigator will use fusions of rhodopsin with green fluorescent protein (GFP). Oligonucleotide effects on transcription will be measured as a decrease in fluorescence intensity; oligonucleotide effects on gene correction and mutation will be measured by the appearance of GFP fluorescence starting with rhodopsin-GFP fusion constructs that are not expressed (GFP-). Cellular studies will precede whole animal experiments in order to test various treatment parameters. Although oligonucleotide-mediated effects on transcription can be measured readily, detection of recombination-based correction and mutational knockout requires specialized constructs, which the investigator will initially test at the selectable adenosine phosphoribosyl transferase (APRT) gene in CHO cells. A sensitive detection system will allow us to detect weak signals that can then be improved and optimized. Using CHO oligonucleotides (TFOs), RNA/DNA chimeric oligonucleotides and oligonucleotide analogues such as peptide nucleic acids (PNAs), for their ability to inhibit transcription, correct gene defects, and introduce mutations. These results will be used as the basis for developing treatment protocols in mice.